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IntroductionThe chronic habit of betel quid chewing has a strong correlation with oral cancer in Myanmar and other Southeast Asian countries (1, 2). In Myanmar, betel quid is betel leaf wrapped with lime and areca nut, with or without tobacco products. Carcinogenesis associated with the betel quid habit is a multistep process, and an accumulation of several mutations causes the progression of cancer (2, 3). p53 mutation is the most commonly identified in human malignancies, including oral squamous cell carcinoma (OSCC) (4-7). The p53 has a direct effect on apoptosis by upregulating Bax expression (8). p16 inhibits the binding of cyclin-dependent kinases (CDKs) 4 and 6 and cyclin D to form a CDK4-cyclin D complex. This complex promotes the phosphorylation of Rb and the release of transcriptional factor, which accelerates the cell cycle (4, 9, 10).The E6 and E7 proteins from high-risk types of HPV cause degradation of p53 and inactivation of Rb (11-13). Thus, increased risk of oral cancer development may be associated with high-risk HPV infection in combination with other established risk factors such as betel quid chewing (14)(15)(16).In this study, we evaluated p53, Bax, Rb, p16
INK4aand HPV DNA expressions and their relations in betel quid chewing associated OSCCs in Myanmar.
Materials and Methods
Tissue sample collectionSamples from 47 previously untreated OSCC patients were collected at the Department of Oral Medicine and Pathology, Institute of Dental Medicine, Yangon, Myanmar, during January 2000 to August 2001. All cases involved the betel quid chewing habit. Twenty-one cases of normal gingival tissues were used as normal control. The biopsy specimens were fixed in 10% buffered formalin solution and embedded in paraffin.
ImmunohistchemistryAn immunohistochemical study was performed on 8 Nishioka et al. p53, Bax, Rb, p16 and HPV in Myanmar OSCC the paraffin sections by streptavidin-biotinimmunoperoxidase staining methods. Four mm sections were deparaffinised in xylen and rehydrated through a graded ethanol series. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in absolute methanol for 30 min, followed by washing in phosphate-buffered saline (PBS). For better detection of p53, Bax, Rb and p16 INK4a , sections were pretreated with microwave oven heating (three cycles of 5 min in 0.01M citrate buffer, pH 6.0). Nonspecific bindings were blocked by incubating the slides in 10% normal rabbit serum for 10 min. After an overnight incubation with the primary antibodies, antip53 (DO-7, Dako, Glostrup, Denmark, 1:75), Bax (B-9, Santa Cruz, CA, USA, 1:50), anti-human retinoblastoma gene product (Rb1, Dako, Glostrup, Denmark, Denmark, 1:50) and p16INK4a /MTS1 (Chemicon, CA, USA, 1:50), secondary biotinylated anti-mouse antibody was applied followed by streptavidin-biotin-peroxidase complex. The color was developed by 3,4-diaminobenzidine (DAB), after which the sections were counterstained with methyl green. For the negative cont...
